Recent advances in plant genetic engineering have opened new doors to engineer plants having improved characteristics or traits, such as, resistance to plant diseases, insect resistance, herbicidal resistance, enhanced stability or shelf-life of the ultimate consumer product obtained from the plants and improvement of the nutritional quality of the edible portions of the plant. Thus, a desired gene (or genes) from a source different than the plant, but engineered to impart different or improved characteristics or qualities, can be incorporated into the plant's genome. This new gene (or genes) can then be expressed in the plant cell to exhibit the new trait or characteristic.
In order to obtain expression of the newly inserted gene in the plant cell, the proper regulatory signals must be present and be in the proper location with respect to the gene. These regulatory signals include a promoter region, a 5′ non-translated leader sequence and a 3′ transcription termination/polyadenylation sequence.
A promoter is a DNA sequence that directs cellular machinery of a plant to produce RNA from the contiguous coding sequence downstream (3′) of the promoter. The promoter region influences the rate, developmental stage, and cell type in which the RNA transcript of the gene is made. The RNA transcript is processed to produce messenger RNA (mRNA) which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5′ non-translated leader sequence is a region of the mRNA upstream of the protein coding region that may play a role in initiation and translation of the mRNA. The 3′ transcription termination/polyadenylation signal is a non-translated region downstream of the protein coding region that functions in the plant cells to cause termination of the RNA transcript and the addition of polyadenylate nucleotides to the 3′ end of the RNA.
It has been shown that certain promoters are able to direct RNA synthesis at a higher rate than others. These are called “strong promoters”. Certain other promoters have been shown to direct RNA production at higher levels only in particular types of cells or tissues and are often referred to as “tissue specific promoters”. In this group, many seed storage protein genes' promoters have been well characterized and widely used, such as the phaseolin gene promoter of Phaseolus vulgaris, the helianthinin gene of sunflower, the β-conglycinin gene of soybean (Chen et al., (1989) Dev. Genet. 10, 112-122), the napin gene promoter of Brassica napus (Ellerstrom et al, (1996) Plant Mol. Biol. 32, 1019-1027), the oleosin gene promoters of Brassica and Arabidopsis (Keddie et al, (1994) Plant Mol. Biol. 24, 327-340; Li, (1997) Texas A&M Ph.D. dissertation, pp. 107-128; Plant et al, (1994) Plant Mol. Biol. 25, 193-205). Another class of tissue specific promoters is described in, U.S. Pat. No. 5,589,583, issued to Klee et al. on Dec. 31, 1996; these plant promoters are capable of conferring high levels of transcription of chimeric genes in meristematic tissues and/or rapidly dividing cells. In contrast to tissue-specific promoters, “inducible promoters” direct RNA production in response to certain environmental factors, such as heat shock, light, hormones, ion concentrations etc. (Espartero et al, (1994) Plant Mol. Biol. 25, 217-227; Gomez-Gomez and Carrasco, (1998) Plant Physiol. 117, 397-405; Holtorf et al, (1995) Plant Mol. Biol. 29, 637-646; MacDowell et al, (1996) Plant Physiol. 111, 699-711; Mathur et al, (1992) Biochem. Biophys. Acta 1137, 338-348; Mett et al, (1996) Transgenic Res. 5, 105-113; Schoffl et al, (1989) Mol. Gen. Genet. 217, 246-253; Ulmasov et al, (1995) Plant Physiol. 108, 919-927).
Promoters that are capable of directing RNA production in many or all tissues of a plant are called “constitutive promoters”. The ideal constitutive promoter should be able to drive gene expression in all cells of the organism throughout its development. Expression of many so-called constitutive genes, such as actin (McDowell et al., (1996) Plant Physiol. 111, 699-711; Wang et al., (1992) Mol. Cell Biol. 12, 3399-3406), and ubiquitin (Callis et al, (1990) J. Biol. Chem. 265, 12486-12493; Rollfinke et al, (1998) Gene 211, 267-276) varies depending on the tissue types and developmental stages of the plant. The most widely used constitutive promoter, the cauliflower mosaic virus 35S promoter, also shows variations in activity in different plants and in different tissues of the same plant (Atanassova et al., (1998) Plant Mol. Biol. 37, 275-285; Battraw and Hall, (1990) Plant Mol. Biol. 15, 527-538; Holtorf et al., (1995) Plant Mol. Biol. 29, 637-646; Jefferson et al., (1987) EMBO J. 6, 3901-3907; Wilmink et al., (1995) Plant Mol. Biol. 28, 949-955). The cauliflower mosaic virus 35S promoter is also described in U.S. Pat. No. 5,106,739. The tissue-specific expression and synergistic interactions of sub-domains of the promoter of cauliflower mosaic virus are discussed in U.S. Pat. No. 5,097,025, which issued to Benfey et al. on Mar. 17, 1992. A Brassica promoter (hsp80) that provides for constitutive expression of heterologous genes in a wide range of tissues and organs is discussed in U.S. Pat. No. 5,612,472 which issued to Wilson et al. on Mar. 18, 1997.
Some constitutive promoters have been used to drive expression of selectable marker genes to facilitate isolation of transformed plant cells. U.S. Pat. No. 6,174,724 B1, issued to Rogers et al. on Jan. 16, 2001, describes chimeric genes which can be used to create antibiotic or herbicide-resistant plants.
Since the patterns of expression of a chimeric gene (or genes) introduced into a plant are controlled using promoters, there is an ongoing interest in the isolation and identification of novel promoters which are capable of controlling expression of a chimeric gene (or genes).